The overall Project goal is to understand how nitric oxide synthase (NOS) isozymes regulate the synthesis of nitric oxide (NO) and thereby its dual biological activities as (i) a diffusible messenger for neurotransmission, long-term potentiation, platelet aggregation and blood pressure regulation, and (ii) a cytotoxic agent for defense against tumor cells and parasites. The inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) isoforms achieve their key functions via an intriguing calcium-regulated electron-transfer mechanism and a unique assembly of at least five cofactors. Each subunit of the NOS dimer has two modules joined by a calmodulin-binding (CaM) hinge region: 1) an oxygenase domain (NOSox) with heme, tetrahydrobiopterin (H4B), and L-Arg binding sites forming the catalytic center for NO production, plus a single structural Zn site at the dimer interface, and 2) a reductase module (NOSred) with NADPH, FAD, and FMN sites supplying electrons to the heme. Systematic characterizations of all three isozymes and individual NOSox, CaM-binding, and NOSred components will address the complex structural biochemistry underlying NOS activity, isozyme specificity, and regulation. Coupled Stuehr, Tamner, and Getzoff group efforts will insure efficient application of unified structure-function studies. Biochemical and mutational characterizations by the Stuehr group will proceed in concert with coupled experimental crystallographic, solution scattering and electron microscopic results plus computational structural analyses by the Getzoff and Tamner groups. As an integrated whole, this project will provide the basis to develop and test hypotheses, and to thereby bridge the growing gap between huge increases in detailed NOS structural and biochemical data and in-depth comprehension of NOS activities. This work focuses on defining conserved and variable isozyme features responsible for 1) catalytic activity and regulation of NOSox, 2) ligand binding to NOSox isozymes, 3) structure and activity of NOSred, and 4) domain interactions in assembled NOS. Designed NOS mutants will be used to experimentally test emerging principles for NOS structure and function. This coordinated structural biochemistry cycle aims to provide a molecular understanding of the activity, inhibition, and regulation of NOS isozymes relevant to important aspects of their biology. These results will furthermore build the essential framework for a unified understanding of NOS relevant to the design of structure-based inhibitors as desirable chemical tools for studying NOS function and as therapeutic agents for stroke, septic shock, and inflammatory damage.